Human Umbilical Cord Blood Cells Ameliorate Motor Deficits in Rabbits in a Cerebral Palsy Model

Abstract

Cerebral palsy (CP) has a significant impact on both patients and society, but therapy is limited. Human umbilical cord blood cells (HUCBC), containing various stem and progenitor cells, have been used to treat various brain genetic conditions. In small animal experiments, HUCBC have improved outcomes after hypoxic-ischemic (HI) injury. Clinical trials using HUCBC are underway, testing feasibility, safety and efficacy for neonatal injury as well as CP. We tested HUCBC therapy in a validated rabbit model of CP after acute changes secondary to HI injury had subsided. Following uterine ischemia at 70% gestation, we infused HUCBC into newborn rabbit kits with either mild or severe neurobehavioral changes. Infusion of high-dose HUCBC (5 × 10(6) cells) dramatically altered the natural history of the injury, alleviating the abnormal phenotype including posture, righting reflex, locomotion, tone, and dystonia. Half the high dose showed lesser but still significant improvement. The swimming test, however, showed that joint function did not restore to naïve control function in either group. Tracing HUCBC with either MRI biomarkers or PCR for human DNA found little penetration of HUCBC in the newborn brain in the immediate newborn period, suggesting that the beneficial effects were not due to cellular integration or direct proliferative effects but rather to paracrine signaling. This is the first study to show that HUCBC improve motor performance in a dose-dependent manner, perhaps by improving compensatory repair processes.

Fig. 1

HUCBC administration improved neurobehavioral scores at day 5 and day 11 in Severe Group compared to saline and media. * p< 0.05, **p< 0.01, ANOVA

Fig. 2

HUCBC administration improved neurobehavioral scores at day 5 and day 11 in the Mild group. * p< 0.05, **p< 0.01, ANOVA

Fig. 3

Low Dose HUCBC administration improved neurobehavioral scores at day 5 and day 11. n=5, p< 0.05 in all 5 measures, ANOVA

Fig. 4

Two-joint analysis of P11 kits. Saline with H-I (mean, black, n=9) had distinctive deficits both in fore and hind limbs; compared to control (blue dashed, n=18) and Low Dose HUCBC treated kits (green, n=4) following E22 H-I.

Fig. 5

Cells were labeled with T2-shortening superparamagnetic iron oxide MRI contrast (Feridex) or T1- shortening contrast Gadofluorine M. Signal change (A) and contrast-to-noise ratio (B) determined on T2-weighted images for Feridex and T1-weighted images for Gadofluorine on agarose gel phantom with serial cell dilutions on 4.7T. Detection of intravenously infused HUCBC on MRI in vivo. Rabbit kits were imaged on 4.7T magnet 24 hours after infusion of media (C, T2-weighted) or Feridex labeled cells (D, T2-weighted) 24 hours or 72 hours (E) after E29 H-I. F.- T1-weighted image of kit infused with Gadofluorine labeled cells 24 hours after E29 H-I. 2.5x10⁶ HUCBC cells were delivered by a PHD 2000 programmable pump. (Harvard Apparatus Holliston, MA).

Fig 7

Detection of intravenously infused HUCBC ex vivo. High resolution imaging with isotopic 50µm voxel resolution on 14.1T magnet. Agarose phantoms without (A) and with Feridex labeled (B) cells. Rectangle ROIs placed on phantoms are shown with high magnification. Note punctate hypo-intensities in panel B, indicating labeled cells. Perfusion fixed brain of E32 kit infused with media (C) or Feridex labeled cells (D). Arrows point to choroid plexus on C and D. Scale bar 1 mm. E .PCR for human DNA showing most of cortex and thalamus samples having some small amounts of human DNA.